Cinamic compounds and derivatives therefrom for the inhibition of histone deacetylase

ABSTRACT

The invention relates to a compound represented by the following formula (I): 
                         
and pharmaceutically acceptable salts, stereoisomers, enantiomers, prodrugs and solvates thereof. The compounds are useful as an agent for enhancing the neurite outgrowth and preventing or treating of diseases associated with HDAC in particular, tumor or cell proliferative diseases. In particular, the compounds of the invention can be used as an agent for anti-cancer, anti-diabetic, and anti-neurodegenerative diseases such as Alzheimer&#39;s disease, Huntington&#39;s disease, Spinocerebellar Ataxias (SCA), and human spinal muscular atrophy (SMA).

FIELD OF THE INVENTION

The present invention relates to novel cinamic compounds which areuseful as agents for the prevention or treatment of diseases associatedwith histone deacetylase (HDAC). They also can be used as agents forenhancing the neurite outgrowth. In particular, they can be used asagents for anti-cancer, anti-diabetic, or anti-neurodegenerativediseases such as Alzheimer's disease, Huntington's disease,Spinocerebellar Ataxias (SCA) and human spinal muscular atrophy (SMA).

BACKGROUND OF THE INVENTION

Eukaryotic DNA is highly organized and packaged in the nucleus. Theorganization and packaging are achieved through the addition ofproteins, including core histones H2A, H2B, H3 and H4, which form acomplex structure, the chromatin, together with DNA. The modification ofcore histones is of fundamental importance to conformational changes ofthe chromatin. The level of acetylation is related to transcriptionactivity, and then the acetylation induces an open chromatinconformation that allows the transcription machinery access topromoters. Histone deacetylase (HDAC) and histone acetyltransferase(HAT) are enzymes that influence transcription by selectivelydeacetylating or acetylating the ε-amino groups of lysine located nearthe amino termini of core histone proteins. HDACs are a family of 18enzymes (isoforms) that may act as master regulators of many diseases,including cancer, because they are involved in the control of geneexpressions. Disruption of HDACs has been linked to a wide variety ofhuman cancers. HDAC enzymes or isoforms appear to be involved in manydifferent types of cancer.

Histone deacetylase (HDAC) inhibitors are emerging as an exciting newclass of potential anticancer agents for the treatment of solid andhematological malignancies. In recent years, an increasing number ofstructurally diverse HDAC inhibitors have been identified; they inhibitproliferation and induce differentiation and/or apoptosis of tumor cellsin cultures and in animal models. HDAC inhibition causes acetylatednuclear histones to accumulate in both tumoral and normal tissues,providing a surrogate marker for the biological activity of HDACinhibitors in vivo. The effects of HDAC inhibitors on gene expressionare highly selective, leading to transcriptional activation of certaingenes such as the cyclin-dependent kinase inhibitor p21^(WAF1/CIP1) butrepression of others. HDAC inhibition results in acetylation of not onlyhistones but also transcription factors such as p53, GATA-1 and estrogenreceptor-alpha. The functional significance of acetylation ofnon-histone proteins and the precise mechanisms whereby HDAC inhibitorsinduce tumor cell growth arrest, differentiation and/or apoptosis arecurrently the focus of intensive research. HDAC inhibitors currently inclinical trials have shown activity and represent a class of molecularlytargeted anti-tumor agents with potential for efficacy based on a novelmechanism of action.

A review article published in Medicinal Research Reviews, Vol. 26, No.4, pp. 397-413, 2006 states that four classes of HDAC inhibitors,short-chain fatty acids, hydroxamic acids, benzamides and cyclicpeptides, had been reported. Hydroxamic acid-based hybrid polarcompounds (HPCs) are HDAC inhibitors, which induce differentiation atmicromolar or lower concentrations (Journal of the National CancerInstitute, Vol. 92, No. 15, Aug. 2, 2000, pp. 1210-1216). U.S. Pat. No.6,174,905, EP 0847992, JP 258863/96, and Japanese Application No.10138957 disclose that benzamide derivatives induce cell differentiationand inhibit HDAC. SNDX-275 (Entinostat) especially disclosed in Example48 of U.S. Pat. No. 6,174,905 has become a candidate for cancertreatment drug. WO 01/38322 discloses additional compounds that serve asHDAC inhibitors. It is reported in Hum Genet, 2006, 120, pp. 101-110that the benzamide M344 up-regulates SMN2 protein expression infibroblast cells derived from SMA patients up to 7-fold after 64 hoursof treatment. It is reported that sodium butyrate ameliorates phenotypicexpression in a transgenic mouse model of spinal and bulbar muscularatrophy (Human Molecular Genetics, 2004, Vol. 13, No. 11, pp.1183-1192). Trichostatin A, a histone deacetylase inhibitor, was foundto induce ubiquitin-dependent cyclin D1 degradation in MCF-7 breastcancer cells (Molecular Cancer 2006, 5:8). U.S. Pat. No. 7,169,801discloses that compounds having the formula of Z-Q-L-M or Z-L-M may beused to inhibit histone deacetylase. U.S. Pat. No. 6,888,027 covers afamily of Sulphonamide HDAC inhibitors including PXD101. European PatentNumber EP 1 301 184 covers the use of valproic acid and derivatives asHDAC inhibitors in the treatment of solid tumors. WO0222577 indicatesthat hydroxamate compounds are inhibitors of histone deacetylase and areuseful aspharmaceuticals for the treatment of proliferative diseases.Moreover, the anti-diabetic activity of HDAC inhibitors is reported inthe FASEB Journal, 2008, Vol. 22, pp. 944-945 and Diabetes, 2008, Vol.57, pp. 860-867.

N,N′-hexamethylene bisacetamide (HMBA) is an effective inducer ofdifferentiation in a number of transformed cell lines. U.S. Pat. Nos.6,087,367 and RE38506 reports that a number of compounds related to HMBAwith polar groups separated by apolar linkages on a molar basis, are asactive or 100 times more active than HMBA. Furthermore, U.S. Pat. No.7,399,787 reports that histone deacetylase inhibitors related to HMBAsuch as suberoylanilide hydroxamide acid (SAHA) have the ability toinduce growth arrest, differentiation and/or apoptosis of tumor cells.In addition, Laurence Catley et al. reports that NVP-LAQ824 (ahydroxamic acid derivative) and NVP-LAQ824 (a derivative of4-aminomethylcinnamic hydroxamic acid) are potent histone deacetylaseinhibitors (Blood, 1 October 2003, Vol. 102, No. 7, pp. 2615-2622).George P et al. reports that LBH589 induces growth inhibition andregression in tumor cell lines by triggering apoptosis and LBH589 is nowbeing tested in phase I clinical trials as an anticancer agent (Blood105(4): 1768-76 Feb. 15, 2005). Other histone deacetylase inhibitorsknown in the art include pyroxamide, M-carboxycinnamic acidbishydroxamide (CBHA), trichostatin A (TSA), trichostatin C,salicylihydroxamic acid (SBHA), azelaic bishydroxamic acid (ABHA),azelaic-1-hydroxamate-9-anilide (AAHA), 6-(3-chlorophenylureido) carpoichydroxamic acid (3C1-UCHA), oxamflatin, A-161906, scriptaid, PXD-101,cyclic hydroxamic acid-containing peptide (CHAP), ITF-2357, MW2796,MW2996, trapoxin A, FR901228 (FK 228 or Depsipeptide), FR225497,apicidin, CHAP, HC-toxin, WF27082, chlamydocin, sodium butyrate,isovalerate, valerate, 4-phenylbutyrate (4-PBA), 4-phenylbutyrate sodium(PBS), arginine butyrate, propionate, butyramide, isobutyramide,phenylacetate, 3-bromopropionate, tributyrin, valproic acid, valproate,CI-994, 3′-amino derivative of MS-27-275, MGCD0103 and Depudecin (U.S.Publication No. 20080242648).

However, there is still a need to develop a new class of HDAC inhibitorsto prevent or treat cancers and other diseases involving HDAC.

SUMMARY OF THE INVENTION

The object of the invention is to provide a group of compoundsrepresented by the following formula (I):

and pharmaceutically acceptable salts, stereoisomers, enantiomers,prodrugs and solvates thereof. The compounds are useful as an agent forenhancing the neurite outgrowth and preventing or treating diseasesassociated with HDAC

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of NBM-HB-OS01(2h) on the inhibition of cellsgrowth of various cancer cell lines. Inhibition of cancer cells wasshown in Rat C6 glioma cells and Human colon cancer HT-29 cells afterthey were treated with various concentrations of NBM-HB-OS01 for 48 hrsand the results are shown in FIGS. 1( a) and (b), respectively. FIG. 1(c) shows the results of a flow analysis of A549 treated with variousconcentrations of NBM-HB-OS01 for 24 hrs. FIG. 1( d) shows thatNBM-HB-OS01 increased p21 gene expression. Rat C6 glioma cells weretreated with NBM-HB-OS01 for 48 hrs and GAPDH served as an internalcontrol. FIG. 1( e) shows the results of treatment of Rat C6 gliomacells with various concentrations of NBM-HB-OS01 for 72 hrs.Accumulation of hyperacetylated histone H3, hyperacetylated histone H4and p21 was detected in a dose-dependent manner by western blotting.FIG. 1( f) shows the results of treatment of Rat C6 glioma cells with 10μg/mL NBM-HB-OS01 for 1, 2, 3 and 4 hrs. The accumulation of acetylatedH3, acetylated H4, acetylated-tubulin and p21 was seen in adose-dependent manner. β-actin was the internal control. FIG. 1( g)shows the results of treatment of Human breast cancer MCF-7 (EstrogenReceptor positive) cells with 10 μg/mL NBM-HB-OS01 for 24 hrs.Overexpression of Gelsolin protein was observed.

FIG. 2 shows the effects of NBM-C-BX-OS01 (4a) on the inhibition ofcells growth of various cancer cell lines. Inhibition of cancer cellsgrowth was shown after treating cell lines with various concentrationsof NBM-C-BX-OS01 in Rat C6 glioma cells, Human breast cancer MCF-7 cellsfor 24 hrs, and Human lung cancer A549 cells for 48 hrs. The results areshown in FIGS. 2( a), (b) and (c), respectively. FIG. 2( d) shows theresults of treatment of MCF-7 cells with 10 μg/mL NBM-C-BX-OS01 for 1,2, 3 and 4 hrs. The expression of acetylated H3, acetylated H4,acetylated-tubulin, and p21 increased in a dose-dependent manner.β-actin was the internal control. FIG. 2( e) shows the results oftreatment of Rat C6 glioma cells with NBM-C-BX-OS01 of 7.5 μg/mL for 6hrs. Overexpression of acetylated tubulin protein was observed.

FIG. 3 shows the effect of NBM-C-BA-OS01 (17a) on the inhibition ofcells growth of three cancer cell lines. Morphology changes of Humanbreast cancer MCF-7 cells and Rat C6 glioma cells in response toNBM-C-BA-OS01 (2.5, 5.0, 7.5 μg/mL) for 72 hrs are shown in FIGS. 3( a)and (b). Hyperacetylation of Histone H3 protein was detected by treatingHuman lung cancer A549 cells for 6 hrs with NBM-C-BA-OS01 of 7.5 μg/mL.The results are shown in FIG. 3( c).

FIG. 4 shows the cell growth inhibition by the treatment of variousNBM-C-BA-OS01 derivatives. Treatment of NBM-C-BA-OS01, NBM-C-BCA-OS01(17d), and NBM-C-BMA-OS01 (17b) of 7.5 μg/mL inhibited the Rat C6 gliomacells growth in 48 hrs.

FIG. 5 shows the effect of NBM-HB-OS01 derivatives on the cell growthinhibition. FIG. 5( a) shows that NBM-C-BA-OS01 (5 μg/mL),NBM-C-BCX-OS01 (4d) (2.5, 5.0 μg/mL), and NBM-C-BMX-OS01 (4b) (2.5, 5.0μg/mL) inhibited the cell growth of Rat C6 glioma cells in 24 hrs. FIG.5( b) shows that treatment of NBM-C-BX-OS01 (7.5 μg/mL), NBM-C-BCX-OS01(2.5, 5.0, 7.5 μg/mL) and NBM-C-BMX-OS01 (2.5, 5.0, 7.5 μg/mL) for 72hrs induced A549 cells growth inhibition in a dose-dependent manner.Similar results can be seen in treatment of Rat C6 glioma cells withNBM-C-BCX-OS01 and NBM-C-BMX-OS01 of various concentrations for 24 hrs,as shown in FIG. 5( c). FIG. 5( d) shows the results of treatment ofHuman glioma Hs683 cells with NBM-C-BX-OS01 (1.25, 2.5, 5.0 μg/mL) for72 hrs and in FIG. 5( e) shows the results of treatment of Humanglioblastoma 05-MG cells with NBM-C-BCX-OS01 (1.0, 2.0, 4.0 μg/mL), andNBM-C-BMX-OS01 (1.0, 2.0, 4.0 μg/mL) for 72 hrs. Cell growth inhibitionwas observed. The treated cells counted by a trypan blue exclusion assayare plotted in FIG. 5( f).

FIG. 6 shows the effects of NBM-HB-OS01 derivatives on the biologicalactivity in various human cancer cell lines. As shown in FIG. 6( a),inhibition of the cell growth of human breast cancer MDA-MB-231 cellswas observed after treating the cells with various concentrations ofNBM-C-BCX-OS01, NBM-C-BMX-OS01, and NBM-C-BFX-OS01 (4c) for 72 hrs. TheNBM-HB-OS01 derivatives significantly inhibited the growth of humanbreast cancer MDA-MB-231 cells. The treated cells counted by a trypanblue exclusion assay are shown in FIG. 6( b). FIGS. 6( c) and (d) showsthe results of treatment of Human lung cancer A549 cells and Humanglioblastoma 05-MG cells with NBM-C-BCX-OS01 and NBM-C-BMX-OS01 for 72hrs, respectively. FIG. 6( e) shows that NBM-C-BCX-OS01 arrested thegrowth of Human glioma Hs683 cells. Human glioma Hs683 cells weretreated with NBM-C-BCX-OS01 (1.0, 2.0, 4.0 μg/mL) for 72 hrs.

FIG. 7 shows the effects of NBM-C-BCX-OS01 and NBM-C-BMX-OS01 onhistones and associated proteins. Human glioma Hs683 cells were treatedwith various concentrations of NBM-C-BCX-OS01 and NBM-C-BMX-OS01 for 72hrs. As shown in FIG. 7( a), the induction of acetylated Hsp90 andgelsolin proteins was detected in a dose-dependent manner, i.e., Hsp90and CTPS proteins decreased in a dose-dependent manner. It can beobserved in FIG. 7( b) that the expression of p21, acetylated tubulin,acetylated Histone H3, and acetylated Histone H4 was significantlyincreased in a dose-dependent manner. SAHA was used as a positivecontrol and β-actin as an internal control.

FIG. 8 shows the effects of: (a) NBM-T-BMX-OS01 (4b), (b) NBM-T-BCX-OS01(4d), (c) NBM-T-BBX-OS01 (4e) and NBM-C-BBX-OS01 (4e), and (d)NBM-I-BCX-OS01 on the inhibition of cell growth (see FIGS. 8( a) to(d)). Human glioma Hs683 cells were grown in the presence of the abovecompounds of various concentrations (1.0, 2.0 and 4.0 μg/mL) for 72hours. The treated cells counted by a trypan blue exclusion assay areplotted in FIG. 8( e).

FIG. 9 shows the effects of (a) NBM-C-BBX-OS01 and NBM-T-BCX-OS01, (b)NBM-T-BBX-OS01 and NBM-I-BCX-OS01, (c) NBM-T-BMX-OS01, (d)NBM-I-BCX-OS01 and NBM-T-BMX-OS01 and (e) NBM-T-BBX-OS01 on theinhibition of human breast cancer MDA-MB-231 cells (see FIGS. 9( a) to(e)). The results of trypan blue exclusion assay are shown in FIG. 9(f).

FIG. 10 shows that (a) NBM-I-BCX-OS01 and NBM-T-BMX-OS01, and (b)NBM-T-BBX-OS01 and NBM-C-BBX-OS01 arrested the human lung cancer A549cells on the S and G2/M phases (see Figs. (a) and (b)). The results weresimilar to those in Human breast cancer MCF-7 cells (see FIGS. 10( c)and, (d)).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel cinamic derivatives, which areuseful as agents for enhancing the neurite outgrowth and preventing andtreating of diseases associated with HDAC, in particular,neurodegenerative diseases, stroke, diabetic, tumor or other cellproliferative diseases. The compounds of the invention are potent ininhibiting growth in cancer cells via differentiation pathway. Inparticular, they can be used as agents for anti-neurodegenerativediseases such as: Alzheimer's disease, Huntington's disease,Spinocerebellar Ataxias (SCA), and human spinal muscular atrophy (SMA).

Compounds of the Invention

Accordingly, the present invention relates to compounds represented bythe following formula (I):

-   wherein-   R₁ is hydrogen, alkyl, alkenyl, C₅₋₆ cycloalkyl, 5-membered or    6-membered unsaturated carbocycle or 5-membered or 6-membered    heterocycle;-   X is C, O, N or S;-   Y is O, NH or O—C₁₋₄alkyl;-   n is an integer of 0 to 10;-   m is an integer of 0 to 5;-   R₂ and R₃ is independently C₁₋₆ alkyl;-   R₄ is C₅₋₆ cycloalkyl or 5-membered or 6-membered unsaturated    carbocycle or heterocycle which may be substituted with halogen,    CF₃, OR₇ or NR₇R₈, wherein R₇ and R₈ are independently hydrogen or    C₁₋₆ alkyl;-   R₅ is OH, NH₂ or C₅₋₆ cycloalkyl, 5-membered or 6-membered    unsaturated carbocycle or heterocycle wherein the cycloalkyl,    carbocycle and heterocycle may be optionally substituted with    halogen, NH₂, NO₂, C₁₋₆ alkoxy, C₁₋₆ alkylthio, OR₇, NR₇R₈ or CF₃;    and-   R₆ is H, C₁₋₁₀alkyl which may be substituted by hydroxy or    C₂₋₁₀alkenyl, or together with R₁ being —C₂H₂—;-   and pharmaceutically acceptable salts, stereoisomers, enantiomers,    prodrugs and solvates thereof.

In the context of the present specification, the term “alkyl” meansstraight or branched hydrocarbon chains. The alkyl is preferably C₁₋₁₀alkyl. Preferably, the carbon number of alkyl is selected from the groupconsisting of 1 to 8; more preferably, it is C₁₋₆ alkyl or C₁₋₄ alkyl.Examples of alkyl groups include methyl (—CH₃), ethyl (—CH₂CH₃), propyl(—CH₂CH₂CH₃), isopropyl (CH₃)₂CH and butyl (—C₄H₉).

In the context of the present specification, the term “alkenyl” meansboth straight and branched chain unsaturated hydrocarbon groups, whereinthe unsaturation is present only as double bonds. According to theinvention, the alkenyl includes one or more double bonds. The alkenyl ispreferably C₂₋₁₆ alkenyl. More preferably, the carbon number of alkenylis selected from the group consisting of 2 to 12. Examples of alkenylgroups include ethenyl (—CH═CH₂), propenyl (—CH═CHCH₃ or —CH₂CH═CH₂),butenyl (—CH₂CH═CHCH₃ or —CH═CHCH₂CH₃ or —CH₂CH₂CH═CH₂),—CH₂CH═C(CH₃)CH₃, —CH₂—CH═CH—CH₂—CH₂—CH═CH—CH₃ and—CH₂—CH═C(CH₃)—CH₂—CH₂—CH═C(CH₃)—CH₃.

In the context of the present specification, the term “cycloalkyl” meansan aliphatic ring (saturated carbocyclic ring). Preferably, the carbonnumber of cycloalkyl is selected from the group consisting of 3 to 8.More preferably, the carbon number of cycloalkyl is selected from thegroup consisting of 5 to 6. Examples of cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In the context of the present specification, the term “unsaturatedcarbocycle” includes a cyclic substituent consisting of carbon atom andhydrogen atom, and the cyclic part is unsaturated cycle, for example,aryl or cycloalkenyl or the like. The term “cycloalkenyl” includesalkenyl which is the cycloalkyl having one or more double bond, forexample, cyclopropenyl (e.g., 1-cyclopropenyl), cyclobutenyl (e.g.,1-cyclobutenyl), cyclopentenyl (e.g., 1-cyclopenten-1-yl,2-cyclopenten-1-yl, and 3-cyclopenten-1-yl), cyclohexenyl (e.g.,1-cyclohexen-1-yl, 2-cyclohexen-1-yl, and 3-cyclohexen-1-yl),cycloheptenyl (e.g., 1-cycloheptenyl), cyclooctenyl (e.g.,1-cyclooctenyl) or the like. Especially, preferred is 1-cyclohexen-1-yl,2-cyclohexen-1-yl, or 3-cyclohexen-1-yl. The term “aryl” includes singleand fused rings wherein at least one ring is aromatic, for example,phenyl, naphthyl and tetrahydronaphthalenyl.

In the context of the present specification, the phrase “5-membered or6-membered heterocycle” refers to a cyclic ring of five or six atoms,wherein at least one atom of the ring is a heteroatom. The 5-membered or6-membered heterocycle can be aromatic or non-aromatic which issaturated or unsaturated. Preferably, the heteroatom is selected from N,O and S. Examples of heterocycle includes, but not limited to, furyl(e.g., 2-furyl, 3-furyl), thienyl (e.g., 2-thienyl, 3-thienyl), pyrrolyl(e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (e.g.,1-imidazolyl, 2-imidazolyl, 4-imidazolyl), pyrazolyl (e.g., 1-pyrazolyl,3-pyrazolyl, 4-pyrazolyl), triazolyl (e.g., 1,2,4-triazol-1-yl,1,2,4-triazol-3-yl, 1,2,4-triazol-4-yl)tetrazolyl (e.g., 1-tetrazolyl,2-tetrazolyl, 5-tetrazolyl), oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl,5-oxazolyl), isoxazolyl (e.g., 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl), thiazolyl (e.g., 2-thiazolyl, 4-thiazolyl, 5-thiazolyl),thiadiazolyl, isothiazolyl (e.g., 3-isothiazolyl, 4-isothiazolyl,5-isothiazolyl), pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl),pyridazinyl (e.g., 3-pyridazinyl, 4-pyridazinyl)pyrimidinyl (e.g.,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), furazanyl (e.g.,3-furazanyl), pyrazinyl (e.g., 2-pyrazinyl), oxadiazolyl (e.g.,1,3,4-oxadiazol-2-yl), 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl,pyrrolidino, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-imidazolinyl,2-imidazolinyl, 4-imidazolinyl, 1-imidazolidinyl, 2-imidazolidinyl,4-imidazolidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl,1-pyrazolidinyl, 3-pyrazolidinyl, 4-pyrazolidinyl, piperidino,2-piperidyl, 3-piperidyl, 4-piperidyl, piperazino, 2-piperazinyl,2-morpholinyl, 3-morpholinyl, morpholino, tetrahydropyranyl or the like.

In the context of the present specification, the term “halogen” meansfluorine, chlorine, bromine and iodine.

In the context of the present specification, the term “pharmaceuticallyacceptable salt” includes those formed with both organic and inorganicacids and bases. Pharmaceutically acceptable acid addition salts includethose formed from mineral acids such as: hydrochloric, hydrobromic,sulphuric, and phosphoric, acid; and organic acids such as: citric,tartaric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic,formic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic,p-toluenesulphonic, benzenesulphonic and isethionic acids.Pharmaceutically acceptable base salts include ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium and salts with organicbases, including salts of primary, secondary and tertiary amines.

In the context of the present specification, the term “prodrug” means acompound which is converted within the body, e.g., by hydrolysis in theblood, into its active form that has medical effects.

In the context of the present specification, the term “solvate” means acomplex comprising the compound of the invention and a solvent in whichthey are reacted or from which they are precipitated or crystallized.

In the context of the present specification, the term “stereoisomers”are isomeric molecules whose atomic connectivity is the same but whoseatomic arrangement in space is different.

In the context of the present specification, the term “enantiomers” arestereoisomers that are nonsuperimposable complete mirror images of eachother, much as one's left and right hands are “the same” but opposite.

According to the invention, the preferred compound of formula (I) of theinvention is selected from the group consisting of the following:

According to the invention, the compounds of formula (I) of theinvention can inhibit HDAC and thus can be used as agents for theprevention or treatment of diseases associated with histone deacetylase(HDAC). In addition, the compounds of the invention significantlyinhibit growth of multiple cancer cell lines, including those of rat C6glioma, human glioblastoma, human breast cancer cells, human leukemiacells, and human melanoma cells. The mechanism for inhibiting the growthof cancer cells may be via differentiation pathway, in particular viainduced differentiation and regulated cell cycle regulator geneexpression, including those of p21 and cyclin B1. In addition, thecompounds of formula (I) of the invention can mediate neuronaldifferentiation of neural stem cells and thus can be used as agentsagainst stroke and anti-neurodegenerative diseases such as Huntington'sdisease and poly-glutamine disease. These compounds also can be used toenhance long-term memory. In addition, the compounds of formula (I) ofthe invention can control whole-body energy balance through theregulation of GLUT4 transcription and therefore provide new therapeutictargets for the treatment of Type 2 diabetes.

For the therapeutic uses of the compounds of the invention, the dosageadministered will, of course, vary with the compound employed, the modeof administration, the treatment desired and the disorder indicated. Thedaily dosage of the compound of formula (I) may be in the range from 1mg/kg to 20 mg/kg. The invention provides the methods of inhibitingHDAC, treating tumor or cell proliferative disease, stroke, diabetic, orneurodegenerative diseases such as Alzheimer's disease, Huntington'sdisease, Spinocerebellar Ataxias (SCA) and human spinal muscular atrophy(SMA) and enhancing the neurite outgrowth in a subject, comprisingadministrating to the subject a therapeutically effective amount of thecompounds of the invention, respectively.

General Synthesis of the Compounds of Formula I of the Invention

The compounds of the present invention can be prepared by anyconventional means. Suitable processes for synthesizing these compoundsare provided in the examples. Generally, compounds of formula (I) can beprepared according to one of the described synthetic schemes below:

General Procedure for the Preparation of 2

To the mixture of 1 (2 g, 8.20 mmol) and potassium t-butoxide (1.84 g,16.4 mmol) in dry DMF (20 mL) was added various benzyl chlorides (16.4mmol), the resulting solution was stirred at room temperature undernitrogen for 6 h and then diluted with EtOAc (50 mL), washed withdis-H₂O (25 mL×3) and dried over Na₂SO₄. After removal of EtOAC underreduced pressure, the residue was purified by silica gel(EtOAc:n-Hexane=1:10˜1:1) to give 2.

2h can be prepared by a similar procedure with reaction temperature of90° C.

(Z)-2-Benzoxy-3-prenyl-4-methoxy-t-butylcinamate (2a)

¹H-NMR (500 MHz, CDCl₃): δ 7.50 (1H, d, J=8.7 Hz), 7.43-7.32 (5H, m),7.06 (1H, d, J=12.4 Hz), 6.67 (1H, d, J=8.7 Hz), 5.82 (1H, d, J=12.4Hz), 5.15 (1H, t, J=6.5 Hz), 4.82 (2H, s), 3.84 (3H, s), 3.35 (2H, d,J=6.7 Hz), 1.70 (3H, s), 1.65 (3H, s), 1.44 (9H, s). (cis)

(Z)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-t-butyl cinamate (2b)

¹H-NMR (500 ^(MHz), CDCl₃): δ 7.50 (1H, d, J=8.6 Hz), 7.35-7.30 (2H, m),7.06 (1H, d, J=12.3 Hz), 6.90 (2H, d, J=8.4 Hz), 6.67 (1H, d, J=8.6 Hz),5.83 (1H, d, J=12.4 Hz), 5.15 (1H, t, J=6.5 Hz), 4.76 (2H, s), 3.86 (3H,s), 3.82 (3H, s), 3.34 (2H, d, J=6.6 Hz), 1.71 (3H, s), 1.65 (3H, s),1.44 (9H, s). (cis)

(Z)-2-Benzoxy-4-methoxy-3-(2-Hydroxy-2-methylbutyl)benzyl cinamate (2h)

1H-NMR (500 MHz, CDCl3): δ 7.35 (1H, d, J=8.6 Hz), 7.25-7.12 (10H, m),7.01 (1H, d, J=12.3 Hz), 6.45 (1H, d, J=8.6 Hz), 5.77 (1H, d, J=12.3Hz), 4.98 (2H, s), 4.66 (2H, s), 3.68 (3H, s), 1.47 (2H, m), 1.04 (6H,s)

General Procedure for the Preparation of 3

The mixture of 2 (11.36 mmol) and 10% KOH/MeOH (40 mL) was refluxedovernight under N₂ and then diluted with dis-H₂O (100 mL), acidifiedwith 2N HCl to pH 5˜6 and extracted with EtOAC (50 mL×3), respectively.The combined EtOAc layer was dried over Na₂SO₄ and concentrated underreduced pressure. The residual was purified by silica gel(EtOAc:n-Hexane=1:2) to give 3.

(Z)-2-Benzoxy-3-prenyl-4-methoxy-cinamate (3a)

¹H-NMR (500 MHz, CDCl₃): δ 7.63 (1H, d, J=8.6 Hz), 7.42-7.26 (5H, m),7.25 (1H, d, J=12.5 Hz), 6.80 (1H, d, J=8.7 Hz), 5.88 (1H, d, J=12.5Hz), 5.16 (1H, t, J=6.6 Hz), 4.82 (2H, s), 3.85 (3H, s), 3.36 (2H, d,J=6.7 Hz), 1.65 (3H, s), 1.62 (3H, s). (cis)

General Procedure for the Preparation of 4

To a solution of potassium hydroxide (637 mg, 11.36 mmol) in MeOH (4 mL)was added hydroxylamine hydrochloride (790 mg, 11.36 mmol) dropwise andthen stirred at ice-bath for 1 h. Filtration to remove the white saltyielded free hydroxylame in MeOH solution. To the mixture of 3a (1 g,2.84 mmol) in dry THF (25 mL) was added ethyl chloroformate (0.6 mL,5.68 mmol) and triethylamine (0.6 mL, 5.68 mmol) and the solution wasstirred at for 0.5 h and then the prepared free hydroxylamine solutionwas added. After reacting for 3 h, the reaction mixture was concentratedunder reduced pressure to give residue. The residue was purified bysilica gel (EtOAc:n-Hexane=1:2) to give 4a.

(Z)-2-Benzoxy-3-prenyl-4-methoxy-N-hydroxy-cinamide (4a)

¹H-NMR (500 MHz, CDCl₃): δ 7.40-7.33 (5H, m), 7.30 (1H, d, J=8.6 Hz),7.01 (1H, d, J=12.5 Hz), 6.66 (1H, d, J=8.7 Hz), 5.79 (1H, d, J=12.4Hz), 5.17 (1H, t. J=6.6 Hz), 4.82 (2H, s), 3.83 (3H, s), 3.37 (2H, d,J=6.7 Hz), 1.69 (3H, s), 1.66 (3H, s); ¹³C-NMR (100 MHz, CDCl₃): δ 159.7(s), 159.2 (s), 155.9 (s), 136.9 (s), 135.9 (d), 131.8 (s), 128.6 (d),128.5 (d), 128.2 (d), 128.1 (d), 128.0 (d), 123.9 (s), 122.7 (d), 121.1(s), 118.2 (d), 106.8 (d), 76.6 (t), 55.7 (q), 25.7 (q), 23.2 (t), 17.9(q), 14.4 (q). (cis)

(Z)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-N-hydroxycinamide (4b)

¹H-NMR (500 MHz, CDCl₃): δ 7.35 (1H, d, J=8.6), 7.29 (2H, d, J=8.5 Hz),6.99 (1H, d, J=12.5 Hz), 6.89 (2H, d, J=8.6 Hz), 6.66 (1H, d, J=8.6 Hz),5.79 (1H, d, J=12.4 Hz), 5.17 (1H, t, J=6.8 Hz), 4.76 (2H, s), 3.83 (3H,s), 3.81 (3H, s), 3.37 (2H, d, J=6.6 Hz), 1.71 (3H, s), 1.67 (3H, s).(cis)

(Z)-2-(4-Fluorobenzoxy)-3-prenyl-4-methoxy-N-hydroxycinamide (4c)

¹H-NMR (500 MHz, CDCl₃): δ 7.40 (1H, d, J=7.8 Hz), 7.36 (2H, d, J=8.5Hz), 7.06 (2H, d, J=8.5 Hz), 7.00 (1H, d, J=12.4 Hz), 6.65 (1H, d, J=7.8Hz), 5.79 (1H, d, J=12.4 Hz), 5.14 (1H, t, J=6.2 Hz), 4.76 (2H, s), 3.80(3H, s), 3.33 (2H, d, J=6.5 Hz), 1.67 (3H, s), 1.65 (3H, s). (cis)

(Z)-2-(4-Chlorobenzoxy)-3-prenyl-4-methoxy-N-hydroxycinamide (4d)

¹H-NMR (500 MHz, CDCl₃): δ 7.34-7.31 (4H, m), 6.99 (1H, d, J=12.2 Hz),6.69 (2H, d, J=8.3 Hz), 5.81 (1H, d, J=12.4 Hz), 5.13 (1H, t, J=6.2 Hz),4.77 (2H, s), 3.85 (3H, s), 3.33 (2H, d, J=6.5 Hz), 1.67 (3H, s), 1.65(3H, s). (cis)

(Z)-2-(4-Bromobenzoxy)-3-prenyl-4-methoxy-N-hydroxycinamide (4e)

¹H-NMR (500 MHz, CDCl₃): δ 7.47 (2H, d, J=8.1 Hz), 7.40 (1H, d, J=8.4Hz), 7.28 (2H, d, J=8.1 Hz), 6.95 (1H, d, J=12.5 Hz), 6.62 (1H, d, J=8.4Hz), 5.76 (1H, d, J=12.4 Hz), 4.73 (2H, s), 3.76 (3H, s), 3.31 (2H, d,J=6.1 Hz), 1.64 (6H, s). (cis)

(Z)-2-(4-Trifluoromethylbenzoxy)-3-prenyl-4-methoxy-N-hydroxycinamide(4f)

¹H-NMR (500 MHz, CDCl₃): δ 7.64 (2H, d, J=8.1 Hz), 7.52 (2H, d, J=8.0Hz), 7.43 (1H, d, J=8.4 Hz), 7.00 (1H, d, J=12.4 Hz), 6.68 (1H, d, J=8.6Hz), 5.80 (1H, d, J=12.4 Hz), 5.14 (1H, t, J=6.4 Hz), 4.86 (2H, s), 3.84(3H, s), 3.41 (2H, d, J=6.6 Hz), 1.66 (3H, s), 1.63 (3H, s). (cis)

(Z)-2-(3,4,5-Trimethoxybenzoxy)-3-prenyl-4-methoxy-N-hydroxycinamide(4g)

¹H-NMR (500 MHz, CDCl₃): δ 7.41 (1H, d, J=8.3 Hz), 6.70 (1H, d, J=12.5Hz), 6.65 (1H, d, J=8.4 Hz), 6.62 (1H, s), 6.61 (1H, s), 5.77 (1H, d,J=12.3 Hz), 5.17 (1H, t, J=6.6 Hz), 4.75 (2H, s), 3.84 (9H, s), 3.80(3H, s), 3.35 (2H, d, J=6.4 Hz), 1.69 (3H, s), 1.65 (3H, s). (cis)

General Procedure for the Preparation of 5

The mixture of 3 (17.04 mmol), HOBT (2.76 g, 20.44 mmol) and DCC (4.22g, 20.44 mmol) in dry THF (30 mL) was stirred at room temperature for0.5 h and then added o-phenylenediamine (1.84 g, 17.04 mmol). Theresulting solution was stirred continuously overnight and thenconcentrated under reduced pressure to obtain the residue. The residuewas dissolved in CH₂Cl₂ (50 ml), washed with saturated NaHCO₃ (25 mL×3)and dried over Na₂SO₄. The organic layer was evaporated under reducedpressure and then purified by silica gel (EtOAc:n-Hexane=1:3˜1:1) togive 5.

(Z)-2-Benzoxy-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide (5a)

¹H-NMR (500 MHz, CDCl₃): δ 7.43-7.38 (5H, m), 7.37 (1H, d, J=8.6 Hz),7.03 (1H, d, J=12.5 Hz), 7.01-7.02 (2H, m), 6.74-6.72 (2H, m), 6.68 (1H,d, J=8.6 Hz), 6.06 (1H, d, J=12.4 Hz), 5.14 (1H, t. J=6.6 Hz), 4.90 (2H,s), 3.84 (3H, s), 3.65 (2H, s), 3.38 (2H, d, J=6.5 Hz), 1.72 (3H, s),1.65 (3H, s). (cis)

(Z)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide(5b)

¹H-NMR (500 MHz, CDCl₃): δ 7.41 (1H, d, J=8.5 Hz), 7.33 (2H, d, J=8.3Hz), 7.00 (1H, d, J=12.9 Hz), 6.88 (2H, d, J=8.4 Hz), 6.73-6.72 (2H, m),6.67 (1H, d, J=8.6 Hz), 6.04 (1H, d, J=12.4 Hz), 5.15 (1H, t, J=6.6 Hz),4.83 (2H, s), 3.87 (3H, s), 3.77 (3H, s), 3.38 (2H, d, J=6.3 Hz), 1.74(3H, s), 1.67 (3H, s). (cis)

(Z)-2-(4-Chlorobenzoxy)-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide(5c)

¹H-NMR (500 MHz, CDCl₃): δ 7.44 (1H, d, J=8.6 Hz), 7.35 (4H, m), 7.12(1H, d, J=12.4 Hz), 7.00 (2H, d, J=10.5 Hz), 6.74 (2H, m), 6.69 (1H, d,J=8.6 Hz), 6.07 (1H, d, J=12.4 Hz), 5.13 (1H, t, J=6.6 Hz), 4.85 (2H,s), 3.84 (3H, s), 3.35 (2H, d, J=6.3 Hz), 1.65 (3H, s), 1.59 (3H, s).(cis)

General Procedure for the Preparation of 6

To a solution of 5 (1 mmol) in THF (15 mL) was added 49% H₂SO₄ (10 mL)and stirred at room temperature for 6 h. The resulting solution wasextracted with CH₂Cl₂ (50 mL×3) and then dried over Na₂SO₄ to giveresidue. The residue was purified by silica gel (EtOAc:n-Hexane=2:1).

(Z)-2-Benzoxy-3-(2-Hydroxy-2-methylbutyl)-4-methoxy-N-(2-aminophenyl)cinamide(6a)

¹H-NMR (500 MHz, CDCl₃): δ 7.43-7.38 (5H, m), 7.37 (1H, d, J=8.6 Hz),7.03 (1H, d, J=12.5 Hz), 7.01-7.02 (2H, m), 6.74-6.72 (2H, m), 6.68 (1H,d, J=8.6 Hz), 6.06 (1H, d, J=12.4 Hz), 4.92 (2H, s), 3.81 (3H, s), 2.68(2H, m), 1.62 (2H, m), 1.18 (6H, s). (cis)

(Z)-2-(4-Methoxybenzoxy)-3-(2-Hydroxy-2-methylbutyl)4-methoxy-N-(2-aminophenyl)cinamide(6b)

¹H-NMR (500 MHz, CDCl₃): δ 7.41 (1H, d, J=8.5 Hz), 7.33 (2H, d, J=8.3Hz), 7.00 (2H, d, J=12.9 Hz), 6.88 (2H, d, J=8.4 Hz), 6.73-6.72 (4H, m),6.67 (1H, d, J=8.6 Hz), 6.04 (1H, d, J=12.4 Hz), 4.83 (2H, s), 3.87 (3H,s), 3.77 (3H, s), 2.67 (2H, m), 1.64 (2H, m), 1.74 (3H, s), 1.67 (3H,s). (cis)

(Z)-2-(4-Chlorobenzoxy)-3-(2-Hydroxy-2-methylbutyl)-4-methoxy-N-(2-aminophenyl)cinamide(6c)

¹H-NMR (500 MHz, CDCl₃): δ 7.44 (1H, d, J=8.6 Hz), 7.35 (4H, m), 7.12(1H, d, J=12.4 Hz), 7.00 (2H, d, J=10.5 Hz), 6.74 (2H, m), 6.69 (1H, d,J=8.6 Hz), 6.07 (1H, d, J=12.4 Hz), 4.85 (2H, s), 3.84 (3H, s), 3.35(2H, d, J=6.3), 2.68-2.64 (2H, m), 1.65 (3H, s), 1.63-1.57 (2H, m), 1.59(3H, s). (cis)

(Z)-2,4-Dimethoxy-3-prenyl cinamate (7)

To a solution of 1 (2.44 g, 10 mmol) and KOH (4.20 g, 75 mmol) dissolvedin EtOH (100 ml) was added MeI (2.51 ml, 40 mmol) dropwise and thenheated to 90° C. for 24 h. The mixture was diluted with dis-H₂O (100ml), acidified with IN HCl to pH 3-4 and then extracted with EtOAc (50ml×3). The combined EtOAc layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by silicagel (EtOAc:n-Hexane=6:1) to obtain 7.

¹H-NMR (500 MHz, CDCl₃): δ 7.62 (1H, d, J=8.6 Hz), 7.23 (1H, d, J=12.5Hz), 6.64 (1H, d, J=8.7 Hz), 5.92 (1H, d, J=12.5 Hz), 5.17 (1H, t, J=6.3Hz), 3.84 (3H, s), 3.71 (3H, s), 3.34 (2H, d, J=6.7 Hz), 1.77 (3H, s),1.67 (3H, s).

(Z)-2,4-Dimethoxy-3-prenyl-N-hydroxy cinamide (8)

Following the procedure similar to that of 4 and further purification bysilica gel (EtOAc:n-Hexane=1:1) gave 8.

¹H-NMR (500 MHz, CDCl₃): δ 7.36 (1H, d, J=8.6 Hz), 7.02 (1H, d, J=12.5Hz), 6.65 (1H, d, J=8.6 Hz), 5.87 (1H, d, J=12.5 Hz), 5.16 (1H, t. J=6.9Hz), 3.82 (3H, s), 3.70 (3H, s), 3.34 (2H, d, J=6.8 Hz), 1.77 (3H, s),1.68 (3H, s).

(Z)-2,4-Dimethoxy-3-prenyl-N-(2-aminophenyl)cinamide (9)

Following the procedure similar to that of 5 and further purification bysilica gel (EtOAc:n-Hexane=2:3) gave 9.

¹H-NMR (500 MHz, CD₃OD): δ 7.48 (1H, d, J=8.6 Hz), 7.09 (1H, d, J=12.4Hz), 7.00-6.97 (2H, m), 6.83 (1H, d, J=8.1 Hz), 6.72-6.70 (2H, m), 6.16(1H, d, J=12.4 Hz), 5.12 (1H, t, J=6.9 Hz), 3.81 (3H, s), 3.73 (3H, s),3.35 (2H, d, J=6.7 Hz), 1.75 (3H, s), 1.63 (3H, s).

(Z)-2-Benzoxy-3-(2-Hydroxy-2-methylbutyl)-4-methoxy-t-butyl cinamate(10)

To a solution of 2a (0.27 mmol) in THF (15 mL) was added 49% H₂SO₄ (10mL) and stirred at room temperature for 6 h. The resulting solution wasextracted with CH₂Cl₂ (50 mL×3) and then dried over Na₂SO₄ to giveresidue. The residue was purified by silica gel (EtOAc:n-Hexane=1:1).

¹H-NMR (500 MHz, CDCl₃): δ 7.38 (1H, d, J=8.7 Hz), 7.36-7.27 (5H, m),7.00 (1H, d, J=12.4 Hz), 6.60 (1H, d, J=8.7 Hz), 5.94 (1H, d, J=12.4Hz), 5.13 (2H, s), 4.81 (2H, s), 3.82 (3H, s), 2.67 (2H, t, J=8.2 Hz),1.61 (2H, t, J=8.2 Hz), 1.44 (9H, s), 1.18 (6H, s).

(Z)-2-Benzoxy-3-(2,3-epoxy-2-methylbutyl)-4-methoxy-t-butyl cinamate(11)

To a solution of 2a (1 g, 2.67 mmol) in CH₂Cl₂(10 ml) was added 70%MCPBA (553 mg, 3.20 mmol) and stirred at rt for 2 h. The reactionmixture was washed with 1NaHSO₃, 10% NaHCO₃ and dried over Na₂SO₄. TheCH₂Cl₂ was evaporated under reduced pressure and then purified by silicagel (EtOAc:n-Hexane=9:1) to obtain 11.

¹H-NMR (500 MHz, CDCl₃): δ 7.55 (1H, d, J=8.6 Hz), 7.43 (2H, d, J=7.1Hz), 7.38-7.32 (3H, m), 7.08 (1H, d, J=12.4 Hz), 6.69 (1H, d, J=8.7 Hz),5.86 (1H, d, J=12.4 Hz), 4.92 (1H, d, J=11.1 Hz), 4.83 (1H, d, J=11.1Hz), 3.86 (3H, s), 2.97-2.94 (2H, m), 2.83-2.79 (1H, m), 1.44 (9H, s),1.33 (3H, s), 1.25 (3H, s).

(Z)-2-Benzoxy-3-(2,3-dihydroxy-2-methylbutyl)-4-methoxy-cinamate (12)

Following the reaction procedure similar to that of 5 and furtherpurification by silica gel (EtOAc:n-Hexane=1:4) gave 12.

¹H-NMR (500 MHz, CDCl₃): δ 7.57 (1H, d, J=8.6 Hz), 7.40-7.34 (5H, m),7.23 (1H, d, J=12.6 Hz), 6.72 (1H, d, J=8.7 Hz), 5.97 (1H, d, J=12.4Hz), 4.91 (1H, d, J=11.1 Hz), 4.81 (1H, d, J=11.1 Hz), 3.88 (1H, t,J=10.5 Hz), 3.85 (3H, s), 2.63 (2H, d, J=10.5 Hz), 1.22 (3H, s), 1.16(3H, s).

(E) 2-Hydroxy-3-prenyl-4-methoxy-ethyl cinamate (13)

To the solution of 1 (2.40 g, 10 mmol) in dry EtOH (20 mL) was added themixture of sodium ethoxide (1.36 g, 20 mmol) in dry EtOH (20 mL)dropwise, the resulting solution was heated under nitrogen for 6 h andthen diluted with dis-H₂O (50 mL), acidified with 1N HCl_((aq)) to pH4-5, extracted with EtOAc (50 mL×3) and dried over Na₂SO₄. After removalof EtOAc under reduced pressure, the residue was purified by silica gel(EtOAc:n-Hexane=10:1) to give 13.

¹H-NMR (500 MHz, CDCl₃): δ 7.92 (1H, d, J=16.1 Hz), 7.33 (1H, d, J=8.7Hz), 6.49 (1H, d, J=8.7 Hz), 6.44 (1H, d, J=16.1 Hz), 6.10 (1H, s), 5.20(1H, t, J=7.0 Hz), 4.23 (2H, q, J=7.2 Hz), 3.82 (3H, s), 3.42 (1H, d,J=7.1 Hz), 1.82 (3H, s), 1.75 (3H, s), 1.31 (3H, t, J=7.2 Hz). (trans)

General Procedure for the Preparation of 14

To the mixture of 13 (1.72 mmol) and K₂CO₃ (4.3 mmol) in acetone (20 mL)was added appropriate benzyl bromide (3.44 mmol), the resulting solutionwas heated under N₂ overnight. After filteration to remove K₂CO₃, thefiltrate was condensed under reducted pressure. The resulting residuewas purified by gel (EtOAc:n-Hexane=15:1) to give 14.

(E)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-ethyl cinamate (14a)

¹H-NMR (500 MHz, CDCl₃): δ 7.98 (1H, d, J=16.1 Hz), 7.44 (1H, d, J=8.7Hz), 7.40 (2H, d, J=8.4 Hz), 6.92 (2H, d, J=8.4 Hz), 6.71 (1H, d, J=8.7Hz), 6.34 (1H, d, J=16.1 Hz), 5.17 (1H, t, J=6.4 Hz), 4.74 (2H, s), 4.25(2H, q, J=7.1 Hz), 3.86 (3H, s), 3.83 (3H, s), 3.39 (2H, d, J=6.5 Hz),1.73 (3H, s), 1.67 (3H, s), 1.33 (3H, t, J=7.1 Hz). (trans)

(E)-2-(4-Chlorobenzoxy)-3-prenyl-4-methoxy-ethyl cinamate (14b)

¹H-NMR (500 MHz, CDCl₃): δ 7.92 (1H, d, J=16.1 Hz), 7.44 (1H, d, J=8.7Hz), 7.41 (2H, d, J=8.4 Hz), 7.37 (2H, d, J=8.4 Hz), 6.72 (1H, d, J=8.7Hz), 6.34 (1H, d, J=16.0 Hz), 5.16 (1H, t, J=6.6 Hz), 4.77 (2H, s), 4.23(2H, q, J=7.2 Hz), 3.86 (3H, s), 3.37 (2H, d, J=6.6 Hz), 1.69 (3H, s),1.67 (3H, s), 1.31 (3H, t, J=7.2 Hz). (trans)

(E)-2-(4-Bromobenzoxy)-3-prenyl-4-methoxy-ethyl cinamate (14c)

¹H-NMR (500 MHz, CDCl₃): δ 7.91 (1H, d, J=16.1 Hz), 7.52 (2H, d, J=8.4Hz), 7.44 (1H, d, J=8.7 Hz), 7.34 (2H, d, J=8.4 Hz), 6.72 (1H, d, J=8.7Hz), 6.34 (1H, d, J=16.0 Hz), 5.16 (1H, t, J=6.6 Hz), 4.75 (2H, s), 4.23(2H, q, J=7.1 Hz), 3.87 (3H, s), 3.36 (2H, d, J=6.6 Hz), 1.70 (3H, s),1.68 (3H, s), 1.31 (3H, t, J=7.1 Hz). (trans)

(E)-2-(3,4,5-Trimethoxybenzoxy)-3-prenyl-4-methoxy-ethyl cinamate (14d)

¹H-NMR (500 MHz, CDCl₃): δ 7.96 (1H, d, J=16.1 Hz), 7.45 (1H, d, J=8.6Hz), 6.72 (1H, d, J=8.8 Hz), 6.70 (2H, s), 6.38 (1H, d, J=16.0 Hz), 5.20(1H, t, J=6.6 Hz), 4.74 (2H, s), 4.22 (2H, q, J=7.1 Hz), 3.90 (6H, s),3.87 (3H, s), 3.86 (3H, s), 3.40 (2H, d, J=6.5 Hz), 1.74 (3H, s), 1.68(3H, s), 1.30 (3H, t, J=7.2 Hz). (trans)

General Procedure for the Preparation of 15

The mixture of 14 (1.81 mmol) and 10% KOH/MeOH (20 mL) was refluxedovernight under N₂ and then diluted with dis-H₂O (100 mL), acidifiedwith 2N HCl to pH 5˜6 and extracted with EtOAc (50 mL×3), respectively.The combined EtOAc layer was dried over Na₂SO₄ and concentrated underreduced pressure to give 15.

(E)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-cinamate (15a)

¹H-NMR (500 MHz, CDCl₃): δ 8.06 (1H, d, J=16.1 Hz), 7.47 (1H, d, J=8.7Hz), 7.39 (2H, d, J=8.5 Hz), 6.93 (2H, d, J=8.5 Hz), 6.73 (1H, d, J=8.7Hz), 6.35 (1H, d, J=16.0 Hz), 5.18 (1H, t, J=6.5 Hz), 4.76 (2H, s), 3.88(3H, s), 3.82 (3H, s), 3.39 (1H, d, J=6.5 Hz), 1.73 (3H, s), 1.68 (3H,s). (trans)

(E)-2-(4-Chlorobenzoxy)-3-prenyl-4-methoxy-cinamate (15b)

¹H-NMR (500 MHz, DMSO-d6): δ 7.69 (1H, d, J=16.0 Hz), 7.64 (1H, d, J=8.8Hz), 7.47 (2H, d, J=9.3 Hz), 7.44 (2H, d, J=9.3 Hz), 6.87 (1H, d, J=8.8Hz), 6.37 (1H, d, J=16.0 Hz), 5.06 (1H, t, J=6.4 Hz), 4.75 (2H, s), 3.82(3H, s), 3.25 (1H, d, J=6.6 Hz), 1.58 (6H, s). (trans)

(E)-2-(4-Bromobenzoxy)-3-prenyl-4-methoxy-cinamate (15c)

¹H-NMR (500 MHz, DMSO-d6): δ 7.71 (1H, d, J=16.1 Hz), 7.65 (1H, d, J=8.7Hz), 7.60 (2H, d, J=8.3 Hz), 7.40 (2H, d, J=8.3 Hz), 6.88 (1H, d, J=8.8Hz), 6.38 (1H, d, J=16.1 Hz), 5.06 (1H, t, J=6.5 Hz), 4.74 (2H, s), 3.82(3H, s), 3.26 (2H, d, J=6.6 Hz), 1.59 (6H, s). (trans)

(E)-2-(3,4,5-Trimethoxybenzoxy)-3-prenyl-4-methoxy-cinamate (15d)

¹H-NMR (500 MHz, CDCl₃): δ 7.96 (1H, d, J=16.1 Hz), 7.45 (1H, d, J=8.6Hz), 6.72 (1H, d, J=8.8 Hz), 6.70 (2H, s), 6.38 (1H, d, J=16.0 Hz), 5.20(1H, t, J=6.6 Hz), 4.74 (2H, s), 3.90 (6H, s), 3.87 (3H, s), 3.86 (3H,s), 3.40 (2H, d, J=6.5 Hz), 1.74 (3H, s), 1.68 (3H, s). (trans)

General Procedure for the Preparation of 16

To a solution of potassium hydroxide (637 mg, 11.36 mmol) in MeOH (4 mL)was added hydroxylamine hydrochloride (790 mg, 11.36 mmol) dropwise andthen stirred in an ice-bath for 1 h. Filtration to remove the white saltgave free hydroxylame in MeOH solution. To the mixture of 15 (2.84 mmol)in dry THF (25 mL) was added ethyl chloroformate (0.6 mL, 5.68 mmol) andtriethylamine (0.6 mL, 5.68 mmol) and stirred for 0.5 h and then addedthe prepared free hydroxylamine solution. After reacting for 3 h, thereaction mixture was concentrated under reduced pressure to giveresidue. The residue was purified by silica gel (EtOAc:n-Hexane=1:1) togive 16.

(E)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-N-hydroxy cinamide (16a)

¹H-NMR (500 MHz, CDCl₃): δ 7.83 (1H, d, J=15.5 Hz), 7.33 (2H, d, J=8.4Hz), 7.27 (1H, d, J=8.6 Hz), 6.88 (2H, d, J=8.3 Hz), 6.52 (1H, d, J=8.6Hz), 6.28 (1H, d, J=15.5 Hz), 5.12 (1H, t, J=5.7 Hz), 4.64 (2H, s), 3.76(3H, s), 3.74 (3H, s), 3.33 (2H, d, J=6.2 Hz), 1.70 (3H, s), 1.64 (3H,s). (trans)

(E)-2-(4-Chlorobenzoxy)-3-prenyl-4-methoxy-N-hydroxy cinamide (16b)

¹H-NMR (500 MHz, MeOH-d4): δ 7.83 (1H, d, J=15.8 Hz), 7.47 (1H, d, J=8.7Hz), 7.44 (2H, d, J=8.2 Hz), 7.37 (2H, d, J=8.3 Hz), 6.81 (1H, d, J=8.7Hz), 6.36 (1H, d, J=15.8 Hz), 5.09 (1H, t, J=6.5 Hz), 4.76 (2H, s), 3.84(3H, s), 3.32 (1H, d, J=6.6 Hz), 1.63 (3H, s), 1.62 (3H, s). (trans)

(E)-2-(4-Bromobenzoxy)-3-prenyl-4-methoxy-N-hydroxy cinamide (16c)

¹H-NMR (500 MHz, acetone-d6): δ 7.86 (1H, d, J=15.8 Hz), 7.60 (2H, d,J=8.4 Hz), 7.51 (1H, d, J=8.7 Hz), 7.49 (2H, d, J=8.3 Hz), 6.86 (1H, d,J=8.7 Hz), 6.50 (1H, d, J=15.8 Hz), 5.14 (1H, t, J=6.7 Hz), 4.80 (2H,s), 3.87 (3H, s), 3.36 (1H, d, J=6.7 Hz), 1.65 (3H, s), 1.61 (3H, s).(trans)

(E)-2-(3,4,5-Trimethoxybenzoxy)-3-prenyl-4-methoxy-N-hydroxy cinamide(16d)

¹H-NMR (500 MHz, CDCl₃): δ 7.88 (1H, d, J=15.7 Hz), 7.35 (1H, d, J=8.6Hz), 6.68 (2H, s), 6.62 (1H, d, J=8.7 Hz), 6.28 (1H, d, J=15.7 Hz), 5.18(1H, t, J=6.0 Hz), 4.68 (2H, s), 3.86 (6H, s), 3.84 (3H, s), 3.82 (3H,s), 3.38 (2H, d, J=6.4 Hz), 1.73 (3H, s), 1.66 (3H, s). (trans)

(E)-2-Benzoxy-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide (17a)

¹H-NMR (500 MHz, CDCl₃): δ 7.87 (1H, d, J=15.1 Hz), 7.40-7.35 (5H, m),7.32 (1H, d, J=7.8 Hz), 7.03 (2H, m), 6.77 (2H, m), 6.67 (1H, d, J=8.1Hz), 6.49 (1H, d, J=15.6 Hz), 5.19 (1H, m), 4.79 (2H, s), 3.85 (3H, s),3.40 (2H, d, J=6.1 Hz), 1.70 (3H, s), 1.67 (3H, s)

(E)-2-(4-Methoxybenzoxy)-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide

¹H-NMR (500 MHz, CDCl₃): δ 7.85 (1H, d, J=15.1 Hz), 7.39 (4H, d, J=7.6Hz), 7.12-7.04 (2H, m), 6.90 (1H, d, J=8.2 Hz), 6.78 (2H, d, J=7.3 Hz),6.68 (1H, d, J=8.1 Hz), 6.54 (1H, d, J=15.2 Hz), 5.19 (1H, m), 4.73 (2H,s), 3.86 (3H, s), 3.77 (3H, s), 3.41 (2H, m), 1.74 (3H, s), 1.67 (3H, s)

(E)-2-(4-Fluorobenzoxy)-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide(17c)

¹H-NMR (500 MHz, CDCl₃): δ 7.90 (1H, d, J=15.2 Hz), 7.43-7.38 (4H, m),7.07-7.03 (4H, m), 6.76 (2H, d, J=7.0 Hz), 6.65 (1H, d, J=8.4 Hz), 6.51(1H, d, J=15.7 Hz), 5.16 (1H, m), 4.76 (2H, s), 3.84 (3H, s), 3.34 (2H,d, J=6.5 Hz), 1.69 (3H, s), 1.66 (3H, s)

(E)-2-(4-Chlorobenzoxy)-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide(17d)

¹H-NMR (500 MHz, CDCl₃): δ 7.88 (1H, d, J=15.4 Hz), 7.39-7.35 (4H, m),7.34 (1H, d, J=7.9), 7.05 (2H, m), 6.78 (2H, m), 6.69 (1H, d, J=8.0 Hz),6.50 (1H, d, J=15.4 Hz), 5.15 (1H, m), 4.78 (2H, s), 3.86 (3H, s), 3.34(2H, d, J=6.3 Hz), 1.68 (3H, s), 1.66 (3H, s)

(E)-2-(4-Bromobenzoxy)-3-prenyl-4-methoxy-N-(2-aminophenyl)cinamide(17e)

¹H-NMR (500 MHz, CDCl₃): δ 7.88 (1H, d, J=15.3 Hz), 7.50 (2H, d, J=7.8Hz), 7.40 (1H, d, J=7.4 Hz), 7.34 (2H, m), 7.05 (2H, m), 6.81 (2H, m),6.71 (1H, d, J=7.6 Hz), 6.52 (1H, d, J=15.5 Hz), 5.15 (1H, m), 4.77 (2H,s), 3.86 (3H, s), 3.37 (2H, m), 1.66 (3H, s), 1.57 (3H, s)

(Z)-3-[6-(4-Chlorobenzoxy)-5-isoprenyloxybenzofuran-5-yl]4-chlorobenzylacrylate (18)

(Z)-3-[6-(4-Chlorobenzoxy)-5-isoprenyloxybenzofuran-5-yl]4-chlorobenzylacrylate (18)

Following the procedure described as 2 gave 18.

¹H-NMR (500 MHz, CDCl₃) δ7.61 (1H, d, J=2.0 Hz), 7.44 (1H, s), 7.36 (2H,d, J=8.3 Hz), 7.31 (2H, d, J=8.3 Hz), 7.23 (2H, d, J=8.3 Hz), 7.18 (1H,d, J=12.3 Hz), 7.11 (2H, d, J=8.3 Hz), 6.66 (1H, d, J=1.9 Hz), 5.99 (1H,d, J=12.3 Hz), 5.58 (1H, t, J=7.1 Hz), 5.06 (2H, s), 5.02 (2H, s), 4.86(2H, d, J=7.1 Hz), 1.78 (3H, s), 1.71 (3H, s).

(Z)-3-[6-(4-Chlorobenzoxy)-5-isoprenyloxybenzofuran-5-yl]acrylate (19)

Following the procedure described as 3 gave 19.

¹H-NMR (500 MHz, CDCl₃) δ7.58 (1H, d, J=2.0 Hz), 7.53 (1H, s), 7.33 (2H,d, J=8.3 Hz), 7.30 (2H, d, J=8.3 Hz), 7.22 (1H, d, J=12.5 Hz), 6.71 (1H,d, J=2.1 Hz), 5.92 (1H, d, J=12.4 Hz), 5.56 (1H, t, J=7.2 Hz), 5.02 (2H,s), 4.85 (2H, d, J=7.1 Hz), 1.75 (3H, s), 1.66 (3H, s).

(Z)-3-[6-(4-Chlorobenzoxy)-5-isoprenyloxybenzofuran-5-yl]-N-hydroxyacrylmide(20)

Following the procedure described as 4 gave 20.

¹H-NMR (500 MHz, CDCl₃) δ7.58 (1H, s), 7.35˜7.30 (4H, m), 7.32 (1H, s),7.00 (1H, d, J=12.4 Hz), ^(˜˜) (1H, s), 5.86 (1H, d, J=12.5 Hz), 5.56(1H, t, J=6.9 Hz), 5.50 (2H, s), 4.89 (2H, d, J=7.1 Hz), 1.76 (3H, s),1.69 (3H, s)

To a solution of 1 (18.51 mmol) in THF (150 mL) was added 49% H₂SO₄ (100mL) and stirred at room temperature for 6 h. The resulting solution wasextracted with CH₂Cl₂ (50 mL×3) and then dried over Na₂SO₄ to giveresidue. The residue was purified by silica gel (EtOAc:n-Hexane=1:2).

¹H-NMR (500 MHz, d₆-acetone): δ 8.02 (1H, d, J=9.5 Hz), 7.91 (1H, d,J=2.1 Hz), 7.41 (1H, s), 6.96 (1H, d, J=2.1 Hz), 6.32 (1H, d, J=9.5 Hz)

Preparation of 8-Methyl-xanthotoxol (22)

Compound 21 (1 g, 4.95 mmol) dissolved in acetone (150 mL). To themixture of the solution of 1 and K₂CO₃ (1.7 g, 12.4 mmol) was addeddimethyl sulfate (0.96 mL, 5.85 mmol), the resulting solution wasrefluxed for 2 hour under nitrogen. After filteration to remove K₂CO₃,the filtrate was condensed under reducted pressure and then diluted withEtOAc (50 mL), washed with dis-H₂O (25 mL×3) and dried over Na₂SO₄.After removal of EtOAc under reduced pressure, the residue was purifiedby silica gel (EtOAc:n-Hexane=1:2) to give 22

8-Methyl-xanthotoxol (22)

¹H-NMR (500 MHz, CDCl₃): δ 7.75 (1H, d, J=9.5 Hz), 7.67 (1H, d, J=5.0Hz), 7.32 (1H, s), 6.80 (1H, d, J=5.0 Hz), 6.35 (1H, d, J=9.5 Hz)

To the solution of 22 (1.06 g, 4.94 mmol) in dry EtOH (25 mL) was addedthe mixture of sodium ethoxide (0.50 g, 7.41 mmol) in dry EtOH (25 mL)dropwise, the resulting solution was refluxed under nitrogen overnightand then diluted with dis-H₂O (50 mL), neutralized with 1N HCl_((aq)) topH 4-5, extracted with EtOAc (50 mL×3) and dried over Na₂SO₄. Afterremoval of EtOAc under reduced pressure, the residue was purified bysilica gel (EtOAc:n-Hexane=2:1) to give 23.

(E)-3-(6-Hydroxy-5-methoxybenzofuran-5-yl)-4-ethyl acrylate (23)

¹H-NMR (500 MHz, CDCl₃) δ8.03 (1H, d, J=16.0 Hz), 7.51 (1H, d, J=2.0Hz), 7.35 (1H, s), 6.68 (1H, d, J=2.0 Hz), 6.63 (1H, d, J=16.5 Hz), 4.29(2H, q, J=7.1 Hz), 4.21 (3H, s), 1.33 (3H, t, J=7.2 Hz).

General Procedure for the Preparation of 24

To the mixture of 23 (1.96 mmol) and K₂CO₃ (4.9 mmol) in acetone (20 mL)was added appropriate benzyl bromide (3.92 mmol), the resulting solutionwas refluxed under nitrogen overnight. After filteration to removeK₂CO₃, the filtrate was condensed under reducted pressure and thendiluted with EtOAc (50 mL), washed with dis-H₂O (25 mL×3) and dried overNa₂SO₄. After removal of EtOAc under reduced pressure, the residue waspurified by silica gel (EtOAc:n-Hexane=1:10) to give 24.

(E)-3-[6-(4-Bromobenzoxy)-5-methoxybenzofuran-5-yl]-4-ethyl acrylate(24a)

¹H-NMR (500 MHz, CDCl₃) δ 8.00 (1H, d, J=16.1 Hz), 7.60 (1H, d, J=2.0Hz), 7.50 (2H, d, J=8.3 Hz), 7.43 (1H, s), 7.35 (2H, d, J=8.3 Hz), 6.72(1H, d, J=2.0 Hz), 6.41 (1H, d, J=16.1 Hz), 5.00 (2H, s), 4.27 (2H, q,J=7.0 Hz), 4.17 (3H, s), 1.34 (3H, t, J=7.1 Hz).

(E)-3-[6-(4-Chlorobenzoxy)-5-methoxybenzofuran-5-yl]-4-ethyl acrylate(24b)

¹H-NMR (500 MHz, CDCl₃) δ 8.00 (1H, d, J=16.0 Hz), 7.61 (1H, d, J=2.5Hz), 7.44 (1H, s), 7.41 (2H, d, J=8.0 Hz), 7.34 (2H, d, J=8.0 Hz), 6.73(1H, d, J=2.0 Hz), 6.41 (1H, d, J=16.0 Hz), 5.02 (2H, s), 4.27 (2H, q,J=7.0 Hz), 4.18 (3H, s), 1.34 (3H, t, J=7.0 Hz).

General Procedure for the Preparation of 25

The mixture of 24 (1.68 mmol) and 10% KOH/MeOH (20 mL) was refluxed for6 hour under N₂ and then diluted with dis-H₂O (100 mL), acidified with2N HCl to pH 2˜3 and extracted with EtOAc (50 mL×3), respectively. Thecombined EtOAc layer was dried over Na₂SO₄ and concentrated underreduced pressure to give 25.

(E)-3-[6-(4-Bromobenzoxy)-5-methoxybenzofuran-5-yl]acrylate (25a)

¹H-NMR (500 MHz, d₆-acetone): δ 8.02 (1H, d, J=16.1 Hz), 7.89 (1H, d,J=2.1 Hz), 7.79 (1H, s), 7.58 (2H, d, J=8.3 Hz), 7.49 (2H, d, J=8.2 Hz),6.92 (1H, d, J=2.1 Hz), 6.50 (1H, d, J=16.0 Hz), 5.10 (2H, s), 4.19 (3H,s)

(E)-3-[6-(4-Chlorobenzoxy)-5-methoxybenzofuran-5-yl]-acrylate (25b)

¹H-NMR (500 MHz, CDCl₃): δ 8.11 (1H, d, J=16.0 Hz), 7.63 (1H, d, J=2.0Hz), 7.48 (1H, s), 7.41 (2H, d, J=8.5 Hz), 7.36 (2H, d, J=8.0 Hz), 6.75(1H, d, J=2.0 Hz), 6.44 (1H, d, J=16.0 Hz), 5.04 (2H, s), 4.19 (3H, s)

General Procedure for the Preparation of 26

To a solution of potassium hydroxide (379 mg, 6.76 mmol) in MeOH (1.8mL) was added hydroxylamine hydrochloride (470 mg, 6.76 mmol) in MeOH(4.7 mL) dropwise for 1 h. Filtration to remove the white salt gave thefree hydroxylame in MeOH solution. To the mixture of 25 (1.69 mmol) indry THF (25 mL) was added ethyl chloroformat (0.3 mL, 2.62 mmol) andtriethylamine (0.4 mL, 6.38 mmol) and stirred at for 0.5 h and thenadded the prepared free hydroxylamine solution under N₂. After reactionfor 2 h, the reaction mixture was diluted with dis-H₂O (100 mL),acidified with 1N HCl to pH 2˜3 and extracted with EtOAc (50 mL×3),respectively. The combined EtOAc layer was dried over Na₂SO₄ andconcentrated under reduced pressure to give residue. The residue waspurified by silica gel (EtOAc:n-Hexane=1:1) to give 26.

(E)-3-[6-(4-Bromobenzoxy)-5-Methoxybenzofuran-5-yl]-N-hydroxyacrylmide(26a)

¹H-NMR (500 MHz, CD₃OD) δ 7.92 (1H, d, J=16.0 Hz), 7.75 (1H, d, J=2.0Hz), 7.50 (2H, d, J=8.0 Hz), 7.47 (1H, s), 7.39 (2H, d, J=8.0 Hz), 6.81(1H, d, J=2.0 Hz), 6.43 (1H, d, J=16.0 Hz), 5.07 (2H, s), 4.14 (3H, s)

(E)-3-[6-(4-Chlorobenzoxy)-5-Methoxybenzofuran-5-yl]-N-hydroxyacrylmide(26b)

¹H-NMR (500 MHz, d₆-acetone) δ 7.96 (1H, d, J=15.8 Hz), 7.83 (1H, d,J=2.0 Hz), 7.53 (2H, d, J=8.1 Hz), 7.52 (1H, s), 7.38 (2H, d, J=8.1 Hz),6.85 (1H, d, J=2.0 Hz), 6.59 (1H, d, J=15.5 Hz), 5.05 (2H, s), 4.15 (3H,s)

8-Methoxymethyl-xanthotoxol (27)

To the mixture of xanthotoxol (21), K₂CO₃ (2.5 eq.) and acetone wasadded MOMCl (2 eq.), the resulting solution was stirred at roomtemperature for 12 h. After removal of acetone, the residue was dilutedwith EtOAc, washed with dis-H₂O and dried over Na₂SO₄. The EtOAC layerwas concentrated under reduced pressure. The residue was subjected tosilica gel chromatography to give 27.

Following the procedure similar to that of 13 gave 28.

General Procedure for the Preparation of 29

Following the procedure similar to that of 14 gave 29.

General Procedure for the Preparation of 30

To the mixture of 29 and MeOH was added 37% HCl(aq), the resultingsolution was stirred at rt for 2 h, then diluted with dis-H2O andextracted with EtOAc. The combined organic layer was dried over Na₂SO₄and concentrated under reduced pressure. The residue was subjected tosilica gel to give 30.

General Procedure for the Preparation of 31

To the mixture of 30, K2CO3 (2.5 eq.) and acetone was added isoprenylbromide (2 eq.), the resulting solution was stirred at room temperaturefor 24 h. After removal of acetone, the residue was diluted with EtOAc,washed with dis-H₂O and dried over Na₂SO₄. The EtOAC layer wasconcentrated under reduced pressure. The residue was subjected to silicagel chromatography to give 31.

General Procedure for the Preparation of 32

Following the procedure described as 3 gave 32.

General Procedure for the Preparation of 33

Following the procedure described as 4 gave 33.

Salts

Pharmaceutically acceptable salts of the compounds of the presentinvention can be prepared by any conventional means. Exemplifiedprocesses for synthesizing the salts are provided below.

(E)-2-(4-Methoxybenzoxy)-4-methoxy-3-prenyl-N-hydroxycinamide lysinesalt

To the mixture of hydroxamate (1 mmole) and EtOH (15 mL) was added asolution of L-lysine (1 mmole) in H₂O (10 mL). The resultant was stirredat 40° C. for 4 h, cooled to room temperature, then stirred foradditional 12 h. The solvent was removed under reduced pressure and theresidue was precipitate from EtOH. After filtration, the solid was washwith dis-H₂O and dried in a vacuum oven.

To the mixture of hydroxamate (1 mmole) and EtOH (15 mL) was added asolution of lithium hydroxide or potassium hydroxide (1.2 mmole) in H₂O(5 mL). The resultant was stirred at room temperature overnight. Thesolvent was removed under reduced pressure and the residue wasprecipitate from EtOH. After filtration, the solid was wash with dis-H₂Oand dried in a vacuum oven.

Pharmaceutical Composition of the Invention

The compounds of formula (I) and pharmaceutically acceptable salts,stereoisomers, enantiomers, prodrugs and solvates thereof may be used ontheir own but will generally be administered in the form of apharmaceutical composition in which the formula (I)compound/salt/solvate (active ingredient) is in association with apharmaceutically acceptable adjuvant, diluent or carrier. Depending onthe mode of administration, the pharmaceutical composition willpreferably comprise from 10 to 30 wt % (percent by weight), morepreferably from 30 to 50 wt %, still more preferably from 50 to 70 wt %,and even more preferably from 70 to 100 wt %, of the active ingredient,all percentages by weight being based on total composition. In addition,the pharmaceutical composition of the invention may further compriseother agents for the prevention or treatment of diseases associated withhistone deacetylase (HDAC).

The pharmaceutical compositions may be administered systemically, e.g.,by oral administration in the form of tablets, capsules, syrups, powdersor granules; or by parenteral administration in the form of solutions orsuspensions; or by subcutaneous administration; or by rectaladministration in the form of suppositories; or transdermally.

The compounds and pharmaceutical compositions of the invention are anHDAC inhibitor and can be retained long in the cells and continuouslyinduce the acetylation of histone H4. They are HDAC inhibitors inducingdifferentiation of cells and neural stem cells. In addition, thecompounds of the invention significantly inhibit HDAC activity. Thecompounds of the invention significantly decrease both S and G2/M phasesof the cells in a dose-dependent manner and change the morphology ofcancer cells. Therefore, the compounds of the invention can treat tumoror cell proliferative disease. Moreover, the compounds of the inventioncan enhance the neurite outgrowth and treat neurodegenerative diseases(such as Huntington's disease and poly-glutamine disease) and humanspinal muscular atrophy (SMA).

EXAMPLE

The following examples illustrate preferred methods for synthesizing andusing the compounds:

Example 1 Preparation of2-(4-Nitrobenzoxy)-4-methoxy-3-prenyl-4-nitrobenzyl cinamate

To the mixture of 1 (2 g, 8.20 mmol) and potassium t-butoxide (1.84 g,16.4 mmol) in dry DMF (20 mL) were added various benzyl chlorides (16.4mmol), and the resulting solution was stirred at room temperature undernitrogen for 6 h and then diluted with EtOAc (50 mL), washed withdis-H₂O (25 mL×3) and dried over Na₂SO₄. After removal of EtOAC underreduced pressure, the residue was purified by silica gel(EtOAc:n-Hexane=1:10˜1:1) to give the title compound: ¹H-NMR (400 MHz,CDCl₃) 8.24-8.22 (4H, m), 7.59-7.46 (4H, m), 7.45 (1H, d, J=8.6 Hz),6.98 (1H, d, J=12.3 Hz), 6.69 (1H, d, J=8.6 Hz), 5.81 (1H, d, J=12.3Hz), 5.12 (1H, t, J=6.5 Hz), 4.91 (2H, s), 4.64 (2H, s), 3.85 (3H, s),3.32 (2H, d, J=6.6 Hz), 1.61 (3H, s), 1.57 (3H, s).

Example 2 Preparation of 2-Benzoxy-4-methoxy-3-prenyl cinamate

The mixture of 2 (11.36 mmol) and 10% KOH/MeOH (40 mL) was refluxedovernight under N₂ and then diluted with dis-H₂O (100 mL), acidifiedwith 2N HCl to pH 5-6 and extracted with EtOAC (50 mL×3). The combinedEtOAc layer was dried over Na₂SO₄ and concentrated under reducedpressure. The residual was purified by silica gel (EtOAc:n-Hexane=1:2)to give the title compound: ¹H-NMR (400 MHz, CDCl₃) 7.63 (1H, d, J=8.6Hz), 7.42-7.26 (5H, m), 7.25 (1H, d, J=12.5 Hz), 6.80 (1H, d, J=8.7 Hz),5.88 (1H, d, J=12.5 Hz), 5.16 (1H, t, J=6.6 Hz), 4.82 (2H, s), 3.85 (3H,s), 3.36 (2H, d, J=6.7 Hz), 1.65 (3H, s), 1.62 (3H, s). ESIMS m/z [M−H]⁻351.13 (100).

Example 3 Preparation of 2-Benzoxy-4-methoxy-3-prenyl-N-hydroxycinnamamide

To a solution of potassium hydroxide (637 mg, 11.36 mmol) in MeOH (4 mL)was added hydroxylamine hydrochloride (790 mg, 11.36 mmol) dropwise, andthen the solution was stirred in an ice bath for 1 h. Filtration wasperformed to remove the white salt to give free hydroxylame in MeOHsolution. To the mixture of 3a (1 g, 2.84 mmol) in dry THF (25 mL) wereadded ethyl chloroformat (0.6 mL, 5.68 mmol) and triethylamine (0.6 mL,5.68 mmol), the mixture was stirred for 0.5 h, and then the preparedfree hydroxylamine solution was added. After reaction for 3 h, thereaction mixture was concentrated under reduced pressure to giveresidual. The residual was purified by silica gel (EtOAc:n-Hexane=1:2)to give the title compound: ¹H-NMR (400 MHz, CDCl₃) 7.40-7.33 (5H, m),7.30 (1H, d, J=8.6 Hz), 7.24 (1H, d, J=8.7 Hz), 7.01 (1H, d, J=12.5 Hz),6.66 (1H, d, J=8.7 Hz), 5.79 (1H, d, J=12.4 Hz), 5.17 (1H, t. J=6.6 Hz),4.82 (2H, s), 3.83 (3H, s), 3.37 (2H, d, J=6.7 Hz), 1.69 (3H, s), 1.66(3H, s); ¹³C-NMR (100 MHz, CDCl₃) 159.7 (s), 159.2 (s), 155.9 (s), 136.9(s), 135.9 (d), 131.8 (s), 128.6 (d), 128.5 (d), 128.2 (d), 128.1 (d),128.0 (d), 123.9 (s), 122.7 (d), 121.1 (s), 118.2 (d), 106.8 (d), 76.6(t), 55.7 (q), 25.7 (q), 23.2 (t), 17.9 (q), 14.4 (q); ESIMS m/z [M+H]⁺368.13 (100).

Example 4 Preparation of2-Benzoxy-4-methoxy-3-(2-Hydroxy-2-methylbutyl)-N-hydroxy cinnamamide

To a solution of 4a (100 mg, 0.27 mmol) in THF (15 mL) was added 49%H₂SO₄ (10 mL) and the solution was stirred at room temperature for 6hours. The resulting solution was extracted with CH₂Cl₂ (50 mL×3) andthen dried over Na₂SO₄ to give residue. The residue was purified bysilica gel (EtOAc:n-Hexane=1:1) to give the title compound: ¹H-NMR (400MHz, CDCl₃) 7.40-7.33 (5H, m), 7.30 (1H, d, J=8.6 Hz), 7.00 (1H, d,J=12.4 Hz), 6.69 (1H, d, J=8.6 Hz), 5.89 (1H, d, J=12.4 Hz), 4.83 (2H,s), 3.82 (3H, s), 2.66 (2H, t, J=7.8 Hz), 1.65 (2H, t, J=7.8 Hz), 1.16(6H, s).

Example 5 Preparation of2-Benzoxy-4-methoxy-3-prenyl-N-(2-aminophenyl)cinamamide

The mixture of 3 (17.04 mmol), HOBT (2.76 g, 20.44 mmol) and DCC (4.22g, 20.44 mmol) in dry THF (30 mL) was stirred at room temperature for0.5 h and then o-phenylenediamine (1.84 g, 17.04 mmol) was added. Theresulting solution was continuously stirred overnight and thenconcentrated under reduced pressure to give residue. The residue wasdissolved in CH₂Cl₂ (50 ml), washed with saturated NaHCO₃ (25 mL×3), anddried over Na₂SO₄. The organic layer was evaporated under reducedpressure and then purified by silica gel (EtOAc:n-Hexane=1:3-1:1) togive the title compound: ¹H-NMR (400 MHz, CDCl₃) 7.43-7.38 (5H, m), 7.37(1H, d, J=8.6 Hz), 7.26 (1H, d, J=8.7 Hz), 7.03 (1H, d, J=12.5 Hz),7.01-7.02 (2H, m), 6.74-6.72 (2H, m), 6.68 (1H, d, J=8.6 Hz), 6.06 (1H,d, J=12.4 Hz), 5.14 (1H, t. J=6.6 Hz), 4.90 (2H, s), 3.84 (3H, s), 3.65(2H, s), 3.38 (2H, d, J=6.5 Hz), 1.72 (3H, s), 1.65 (3H, s).

Example 6 Preparation of2-Benzoxy-4-methoxy-3-(2-Hydroxy-2-methylbutyl)-benzyl cinamamate

To a solution of 2 (0.27 mmol) in THF (15 mL) was added 49% H₂SO₄ (10mL) and the solution was stirred at room temperature for 6 h. Theresulting solution was extracted with CH₂Cl₂ (50 mL×3) and then driedover Na₂SO₄ to give residue. The residue was purified by silica gel(EtOAc:n-Hexane=1:1) give the title compound: ¹H-NMR (400 MHz, CDCl₃)7.38 (1H, d, J=8.7 Hz), 7.36-7.27 (10H, m), 7.00 (1H, d, J=12.4 Hz),6.60 (1H, d, J=8.7 Hz), 5.94 (1H, d, J=12.4 Hz), 5.13 (2H, s), 4.81 (2H,s), 3.82 (3H, s), 2.67 (2H, t, J=8.2 Hz), 1.61 (2H, t, J=8.2 Hz), 1.18(6H, s); ESIMS m/z [M+Na]⁺ 483.6 (100).

Example 7 Inhibition of Cancer Cell Growth by the Compounds of theInvention

Three cancer cell lines, Rat C6 giloma cells, Human breast cancer MCF-7cells, and Human lung cancer A549 cells, were cultured in Dulbecco'smodified Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin. All three cell lines weremaintained at 37° C. in a humidified atmosphere of 95% air and 5% CO₂.For the experiments, the cells were seeded in 6-well plates. After 24hours, the cells were treated with different concentrations of thevarious compounds. The cells were observed at 24, 48, and 72 hours.Inhibition of cancer cell growth of NBM-HB-OS01 in variousconcentrations after 48 hours in Rat C6 glioma cells (see FIG. 1( a)) orin Human colon cancer HT-29 cells (see FIG. 1( b)) was shown.NBM-C-BX-OS01 arrested the cell growth in various concentrations in RatC6 glioma cells in 48 hours (see FIG. 2( a)), in human breast cancerMCF-7 cells in 24 hours (see FIG. 2( b)) and in human lung cancer A549cells in 48 hours (see FIG. 2( c)). Treatments with NBM-C-BA-OS01,NBM-C-BCA-OS01 and NBM-C-BMA-OS01 inhibited the Rat C6 glioma cellgrowth in a fixed concentration (7.5 μg/mL) in 48 hours (see FIG. 4).NBM-C-BA-OS01 (5 μg/mL), NBM-C-BCX-OS01 (2.5, 5.0 μg/mL), andNBM-C-BMX-OS01 (2.5, 5.0 μg/mL) inhibited the cell growth of Rat C6glioma cells in 24 hours (see FIG. 5( a)).

Example 8 Inhibition of Cancer Cell Growth and Change of the Morphologyby the Compounds of the Invention

Five cancer cell lines, Rat C6 giloma cells, Human breast cancer MCF-7cells, Human glioma Hs683 cells, Human glioblastoma 05-MG cells, andHuman lung cancer A549 cells, were cultured in Dulbecco's modifiedEagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS) and1% penicillin/streptomycin, and were kept at 37° C. in a humidifiedatmosphere of 95% air and 5% CO₂. Human breast cancer MDA-MB-231 cellswere cultured in L-15 medium (Gibco) containing 10% fetal bovine serum(FBS), 1% penicillin/streptomycin, and 2 mM glutamine, and weremaintained at 37° C. in a humidified atmosphere of 95% air and 0% CO₂.For these experiments, the cells were seeded in 6-well plates or a 60-mmdish. After 24 hours, the cells were treated with differentconcentrations of the various compounds. The cells were observed at 24,48 and 72 hours. Human breast cancer MCF-7 cells (see FIG. 3( a)), andRat C6 glioma cells (see FIG. 3( b)) exhibited changes in morphology inresponse to NBM-C-BA-OS01 2.5, 5.0, 7.5 μg/mL treatment for 72 hours.Treatments with NBM-C-BX-OS01 (7.5 μg/mL), NBM-C-BCX-OS01 (2.5, 5.0, 7.5μg/mL) and NBM-C-BMX-OS01 (2.5, 5.0, 7.5 μg/mL) induced A549 cell growthinhibition in dose-dependent amounts for 72 hours (see FIG. 5( b)) andthe results were similar to those in Rat C6 glioma cells for 24 hours(see FIG. 5( c)). Human glioma Hs683 cells were treated withNBM-C-BX-OS01 (1.25, 2.5, 5.0 μg/mL) for 72 hours (see FIG. 5( d)).Human glioblastoma 05-MG cells were treated with NBM-C-BCX-OS01 (1.0,2.0, 4.0 μg/mL) and NBM-C-BMX-OS01 (1.0, 2.0, 4.0 μg/mL) for 72 hours(see FIG. 5( e)). The results obtained by cell counting showed the sametendency (see FIG. 5( f)). NBM-C-BCX-OS01, NBM-C-BMX-OS01 andNBM-C-BFX-OS01 inhibited the growth of human breast cancer MDA-MB-231cells and changed the morphology of the cells for 72 hours (see FIG. 6(a)). The results were counted by a trypan blue exclusion assay (see FIG.6( b)).

Example 9 Effects of the Invention (NBM-HB-OS01) on mRNA Expression ofRat C6 Glioma Cells

The cell-cycle related mRNA expression was examined by RT-PCR. The totalRNA was isolated from the treated Rat C6 glioma cells using the RNeasyMini Kit (Qiagen) as described by the manufacturer. The cDNA wasproduced from 500 ng of total RNA using ReverTra-Plus-TM (TOYOBO). TheRT product (1 μl) was amplified by PCR with primers for amplification ofseveral genes (p21, cyclin B1, and cyclin D1) for the cell cycleanalyses and GAPDH was used as an internal control. Rat C6 glioma cellswere treated with NBM-HB-OS01 for 48 hours, and as shown in FIG. 1( d),NBM-HB-OS01 induced p21 mRNA expression.

Example 10 Effects of the Invention on the Cell Cycle of Various HumanCancer Cells

Human lung cancer A549 cells, Human glioma Hs683 cells and Humanglioblastoma 05-MG cells were seeded 1×10⁶ cells in a 100-mm dish. After24 hours of incubation in DMEM+10% BSA, Human lung cancer A549 cellswere treated with NBM-HB-OS01 (2.5, 5.0, 7.5, 10.0 μg/mL) for 24 hoursand were treated with NBM-C-BCX-OS01 (2.5, 5.0, 7.5 μg/mL) andNBM-C-BMX-OS01 (2.5, 5, 7.5 μg/mL) for 72 hours. Human glioblastoma05-MG cells were treated with NBM-C-BCX-OS01 (1.0, 2.0, 4.0 μg/mL) andNBM-C-BMX-OS01 (1.0, 2.0, 4.0 μg/mL) for 72 hours. Human glioma Hs683cells were treated with NBM-C-BCX-OS01 (1.0, 2.0, 4.0 μg/mL). For cellcycle analysis, cells were fixed in 80% ethanol for 1 hour (orovernight) at −20° C. and then incubated with 2 μg/mL RNase A for 30minutes at 37° C. Cells were stained with propidium iodide (5 g/mL PI)in PBS and analyzed using a Becton Dickinson flow cytometer, BD FACScanand CellQuest acquisition and analysis programs. The results of FIG. 1(c) show that NBM-HB-OS01 arrested the human lung cancer A549 cells onthe G0/G1 phase in a dose-dependent manner. Human lung cancer A549 cellswere inhibited by various concentrations (2.5, 5, 7.5 μg/mL) ofNBM-C-BCX-OS01 and NBM-C-BMX-OS01 for 72 hours (see FIG. 6( c)) andHuman glioblastoma 05-MG cells were inhibited by various concentrations(1.0, 2.0, 4.0 μg/mL) of NBM-C-BCX-OS01 and NBM-C-BMX-OS01 for 72 hours(see FIG. 6( d)). Human glioma Hs683 cells were treated withNBM-C-BCX-OS01 (1.0, 2.0, 4.0 μg/mL) for 72 hours. NBM-C-BCX-OS01 couldarrest the growth of Human glioma Hs683 cells (see FIG. 6( e)).

Example 11 Up-Regulation of the HDAC Associated Proteins was Observedafter Treatment of the Compounds of the Inventions

Rat C6 glioma cells, Human breast cancer MCF-7 cells, and Human lungcancer A549 cells were seeded in 6-well plates. After 24 hrs ofincubation in medium+10% BSA, Human breast cancer MCF-7 cells weretreated with NBM-HB-OS01 (10.0 μg/mL) and vorinostat (SAHA, 5 μM) for 24hours. Rat C6 glioma cells were treated with NBM-C-BX-OS01 (7.5 μg/mL)and vorinostat (SAHA, 5 μM) for 6 hours. Human lung cancer A549 cellswere treated with NBM-C-BA-OS01 (7.5 μg/mL) and vorinostat (SAHA, 5 μM)for 6 hours. The treated cells were fixed in 80% methanol for 30 minutesand then washed 3 times with PBS solution. Cells were permeabilized with0.3% Triton X-100 for 30 minutes, and then blocked in 10% fetal bovineserum (FBS) in PBS-T (0.1% Twin 20 in PBS) for 1 hour. The treated cellswere detected with primary antibody against acetyl-Histone H3,acetyl-tubulin, and Gelsolin. Photographs were taken with a Nikonmicroscope. The results indicated that the compounds of the inventioncould induce the HDAC associated proteins expression of the variouscancer cells (see FIG. 1( g), FIG. 2( e), and FIG. 3( c)).

Example 12 Increased Accumulation of Hyperacetylated Histone and Tubulinand p21 in Various Cell Lines Treated with the Compound of the Invention

Rat C6 glioma cells and Human breast cancer MCF-7 cells were seeded5×10⁵ cells in a 60-mm dish or 1×10⁶ cells in a 100-mm dish. After 24hours, Rat C6 glioma cells were treated with various concentrations ofNBM-HB-OS01 (2.5, 5.0, 7.5, 10.0 μg/mL) for 72 hours and were treatedwith 10.0 μg/mL NBM-HB-OS01 for 1, 2, 3, and 4 hours. Human breastcancer MCF-7 cells were treated with 7.5 μg/mL NBM-C-BX-OS01 for 1, 2,3, and 4 hours. Lysates of C6, and MCF-7 cells were prepared for theimmunoblotting of acetyl-Histone H3 (Cell Signaling Technology, Inc.),acetyl-Histone H4 (Upstate), acetyl-tubulin (Sigma Chemical Co.), p21(BD Pharmingen Technology, Inc.), and actin (Sigma Chemical Co.).Proteins were detected by chemiluminescence (ECL, Amersham). The resultsindicated that the accumulation of hyperacetylated histone H3,hyperacetylated histone H4, acetylated-tubulin, and p21 were induced inRat C6 glioma and MCF-7 cells (see FIG. 1( e), FIG. 1( f), and FIG. 2(d)). β-actin is an internal control.

Example 13 Effects of NBM-C-BCX-OS01 and NBM-C-BMX-OS01 on Histones andthe HDAC Associated Proteins

Human glioma Hs683 cells were seeded at 5×10⁵ cells in a 60-mm dish or1×10⁶ cells in a 100-mm dish. After 24 hours, Human glioma Hs683 cellswere treated with different doses of NBM-C-BCX-OS01, NBM-C-BMX-OS01(1.0, 2.0, 4.0 μg/mL), and vorinostat (SAHA, 5 μM) for 72 hours. Lysatesof Hs683 cells were prepared for the immunoblotting of acetyl-Histone H3(Cell Signaling Technology, Inc.), acetyl-Histone H4 (Upstate),acetyl-tubulin (Sigma Chemical Co.), p21 (BD Pharmingen Technology,Inc.), CTPS (ABNOVA TAIWAN Corporation), Gelsolin (Sigma Chemical Co.),Hsp90 (Cell Signaling Technology, Inc.), acetyl-Hsp90 (ROCKLAND, Inc.)and actin (Sigma Chemical Co.). Proteins were detected bychemiluminescence (ECL, Amersham). The increase of acetylated Hsp90 andgelsolin proteins was observed in a dose-dependent manner. Hsp90 andCTPS proteins were decreased in a dose-dependent manner (see FIG. 7(a)). The expression of p21, acetylated tubulin, acetylated Histone H3,and acetylated Histone H4 was induced in a dose-dependent manner. SAHAwas used as a positive control and β-actin as an internal control (seeFIG. 7( b)).

1. A compound represented by the following formula (I):

wherein R₁ is hydrogen, alkyl, alkenyl, C₃₋₈ cycloalkyl, 5-membered or6-membered unsaturated carbocycle or 5-membered or 6-memberedheterocycle; X is C, O, N or S; Y is O, NH or O—C₁₋₄alkyl; n is aninteger of 0 to 10; m is an integer of 1 to 5; R₂ and R₃ isindependently C₁₋₆ alkyl; R₄ is C₅₋₆ cycloalkyl or 5-membered or6-membered unsaturated carbocycle or heterocycle which may besubstituted with halogen, CF₃, OR₇ or NR₇R₈, wherein R₇ and R₈ areindependently hydrogen or C₁₋₆ alkyl; R₅ is OH, NH₂ or C₅₋₆ cycloalkyl,5-membered or 6-membered unsaturated carbocycle or heterocycle whereinthe cycloalkyl, carboycle and heterocycle may be optionally substitutedwith halogen, NH₂, NO₂, C₁₋₆ alkoxy, C₁₋₆ alkylthio, OR₇, NR₇R₈ or CF₃;and R₆ is H, C₁₋₁₀alkyl which may be substituted by hydroxy orC₂₋₁₀alkenyl, or together with R₁ being —C₂H₂—; and pharmaceuticallyacceptable salts, stereoisomers, and enantiomers thereof.
 2. Thecompound according to claim 1, wherein R₁, R₂ and R₃ are independentlyC₁₋₄ alkyl; R₄ is phenyl or phenyl substituted with halogen, CF₃, OC₁₋₄alkyl, R₅ is OH, phenyl or phenyl substituted with NH₂ and R₆ ishydrogen.
 3. The compound according to claim 1, wherein R₁, R₂ and R₃are independently methyl, R₄ is phenyl or phenyl substituted with F, Cl,Br, CF₃, OCH₃, R₅ is OH or phenyl substituted with NH₂ and R₆ ishydrogen.
 4. The compound according to claim 1, wherein X is carbon. 5.The compound according to claim 1, wherein n and m is
 1. 6. The compoundaccording to claim 1, wherein the carbocycle is cycloalkenyl or aryl. 7.The compound according to claim 6, wherein the cycloalkenyl is selectedfrom the group consisting of cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl and cyclooctenyl, and the aryl is selectedfrom the group consisting of phenyl, naphthyl andtetrahydronaphthalenyl.
 8. The compound according to claim 1, whereinthe 5-membered or 6-membered unsaturated heterocycle is selected fromthe group consisting of furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl,triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl,isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, furazanyl, pyrazinyl,oxadiazolyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidino,2-pyrrolidinyl, 3-pyrrolidinyl, 1-imidazolinyl, 2-imidazolinyl,4-imidazolinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 1-pyrazolidinyl,3-pyrazolidinyl, 4-pyrazolidinyl, piperidino, 2-piperidyl, 3-piperidyl,4-piperidyl, piperazino, 2-piperazinyl, 2-morpholinyl, 3-morpholinyl,morpholino and tetrahydropyranyl.
 9. The compound according to claim 1,which is selected from the group consisting of:


10. A pharmaceutical composition comprising the compound of claim 1 orpharmaceutically acceptable salts, stereoisomers, and enantiomersthereof as an active ingredient and a pharmaceutically acceptablecarrier.